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A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.
Maji, Basudeb; Gangopadhyay, Soumyashree A; Lee, Miseon; Shi, Mengchao; Wu, Peng; Heler, Robert; Mok, Beverly; Lim, Donghyun; Siriwardena, Sachini U; Paul, Bishwajit; Dancík, Vlado; Vetere, Amedeo; Mesleh, Michael F; Marraffini, Luciano A; Liu, David R; Clemons, Paul A; Wagner, Bridget K; Choudhary, Amit.
Afiliação
  • Maji B; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.
  • Gangopadhyay SA; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.
  • Lee M; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
  • Shi M; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.
  • Wu P; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.
  • Heler R; Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.
  • Mok B; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
  • Lim D; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
  • Siriwardena SU; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Paul B; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.
  • Dancík V; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Vetere A; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Mesleh MF; Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Marraffini LA; Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA; Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 11231, USA.
  • Liu DR; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
  • Clemons PA; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Wagner BK; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Choudhary A; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA. Electronic address
Cell ; 177(4): 1067-1079.e19, 2019 05 02.
Article em En | MEDLINE | ID: mdl-31051099
ABSTRACT
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ensaios de Triagem em Larga Escala / Sistemas CRISPR-Cas / Proteína 9 Associada à CRISPR Idioma: En Ano de publicação: 2019 Tipo de documento: Article
Texto completo
Imprimir
XML
PubMed Links
Buscar no Google

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ensaios de Triagem em Larga Escala / Sistemas CRISPR-Cas / Proteína 9 Associada à CRISPR Idioma: En Ano de publicação: 2019 Tipo de documento: Article